biology essay

Dna Replication And Cell Growth Cycle Biology Essay

Published: 23, March 2015

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In this study, we analyzed basic cell culture technique and observed the cells under a microscope. We also used two different techniques to count cells. These experiments were done in order to analyze methods to count cells over time and determine their growth rates. The fibroblast cells used were cultured using sterile techniques and under a biohazard hood with laminar flow. Healthy and viable cells were counted using two basic techniques. One of the techniques was using a haemocytometer to count cells and calculate a value for a unit of volume. The other technique used was Vi-CELL counter. This counter counts cells. It distinguishes between live and dead cells because the cells are place in trypan blue. The live and dead cells are reported by a viability number. Both methods produced the exact same cell count so it is assumed that both methods were fairly accurate. Finally, cell count statistics were analyzed. These statistics were analyzed in order to determine cell growth rates and calculate how long cell populations take to double. The cell populations in media B doubled at a much faster rate than the cell populations in media B. This is useful to know to understand cell behavior and what factors can promote or inhibit cell growth.

The cell cycle is a process that occurs constantly throughout our bodies. The whole process is regulated by Cyclin-dependent-kinases or CDKs [3]. The process is composed of four main sections [4]. S phase is the synthesis phase. This is the stage where DNA replication occurs [4]. DNA replication is the process of increasing the amount of genetic material in a cell [1]. This will provide the cell with enough genetic material to support two cells when division occurs. DNA replication requires a protein called DNA helicase to unzip a double helix DNA molecule [2]. Once the molecule is unzipped, complementary base pairs are brought to each individual DNA strand. The end result of DNA replication will be two identical DNA molecules [2]. This process occurs continuously during the S phase of cell growth. The S phase takes up about half of the time during the cell cycle [4]. It can last from 12 to 14 hours [4]. The G1 phase takes place between mitosis and the S phase [4]. This is a phase that is used for growth of each cell. During this phase the cell will increase the amount of proteins and mass [4]. The G2 phase is similar. This phase is also used for growth and it occurs between the S phase and mitosis [4]. These three phases combined, also known as interphase, take up the majority of the cell cycle process. On a 24 hour scale, they would take up 23 hours [4]. Over the remaining one hour, mitosis occurs [4]. Mitosis is also composed of several stages. The first phase of mitosis is called prophase. During this phase, the replicated chromosomes condense [6]. Eventually in prophase, the nuclear envelope begins to break down [6]. It will be completely gone at the end of prophase and the DNA will be dispersed throughout the cell [6]. The next stage is prometaphase. During this stage, microtubules begin to come in contact with the genetic material [6]. The next stage is metaphase. In metaphase, the genetic material attaches to the microtubules and lines up in the center of the cell [6]. Anaphase is the next stage of mitosis. During anaphase, the condensed genetic material is split at the centromeres. Each half begins to go to opposite ends of the cell [6]. The final stage of mitosis is called telophase. This is when the genetic material is at opposite ends of the cell, and the nuclear membrane reforms [6]. After this occurs, the cell will split down the middle in cytokinesis and two identical daughter cells will remain [6]. About half of the cells are in the synthesis phase. Close to half are in the G1 and G2 phases. The remaining cells are undergoing cell division.

Materials and methods [7]:

L292 Harvest

It should be noted that all of the liquids used in the experiment were heated to 37 degrees Celsius prior to use. The parent flask of cells was observed under a phase contrast microscope. This is to ensure the cells are viable and healthy. These murine L929 fibroblast cells should resemble a cobblestone pattern. The observations were recorded. Using careful sterile techniques, the media was poured from the parent flask leaving only the adhered monolayer of fibroblasts. The adhered monolayer of cells was washed twice with 1-2 mL of calcium and magnesium free Dulbecco's phosphate buffered saline (DPBS). This will remove residual media that could block enzyme reactions. 3 mL of Trypsin/Versene (TV) enzyme solution were added to the monolayer. The flask was constantly tilted from side to side to ensure that the TV covered the whole surface. The flask was incubated at 37 degrees Celsius for 5 minutes. After the incubation, the flask was placed under a microscope. The cells needed to be detached from the surface. If they were not detached, the side of the flask was given a sharp tap. If necessary, further incubation could have been necessary. 6 mL of complete medium (EMEM + 10% Foetal Bovine Serum) was added to the flask. This halts trypsinization. The flask suspension was flushed in and out of a 10 mL pipette to break up the cell clumps. The suspension was pipetted into a 10 mL sterile centrifuge tube. The tube was centrifuged for 3 minutes at 1000 rpm. Next, the medium was poured out of the tube. 8 mL of fresh medium was added to the centrifuge tube. The cells were now ready to be counted. This was not done by us but the cells would normally be passaged. In order to do this the cells should be diluted in order to yield 1.5 X 104, 7.5 X 104, or 1.5 X 105 cells in 5 mL of fresh medium. These flasks will achieve confluence in one week. When the subculturing takes place, the new flasks should be labeled with passage number, cell type, date, and initials of the person doing the subculture. The culture flasks would be returned to the incubator.

Cell Counting

Haemocytometer

A haemocytometer was cleaned with 70% ethanol and it was wiped down with lint-free tissues. A coverslip was also washed with 70% ethanol and wiped down with lint-free tissues. A coverslip was placed over the central section of the haemocytometer.

Figure 1: The coverslip should be place over the haemocytometer with no bubbles underneath [7]

The cell suspension was pipetted up and down using a plastic pipette. A small amount of suspension was drawn out into the tip of the pipette and the tip of the pipette was placed onto the haemocytometer where the coverslip meets the central area. The suspension was pipette out on to this edge and it ran onto the slide by capillary action.

Figure 2: This figure shows the method by which the suspension is added to the haemocytometer [7]

The haemocytometer was placed under a microscope and a 10x objective focus was used to observe the result. The grid of the haemocytometer was now visible. The cells were counted in a way to avoid double counting. Cells that crossed or touched the top or the right side of the square were included in the square's count. Cells that were touching the left and bottom of the square were not included in the square's count. The haemocytometer is divided into 9 sections. The cells in the top left section and the bottom right section were counted. The total count was divided by the number of large shaded squares counted (see figure 3). This gave us an average number of cells per square.

Figure 3: In this study, the top left and bottom right sections were counted [7]

Vi-Cell Counter

The Vi-CELL was prepared by the demonstrators. The cell suspension was thoroughly mixed with no signs of cell sedimentation. 0.5 mL of the suspension was transferred to Vi-CELL sample cup. The cup was placed into the Vi-CELL carousel. On the software graphical user interphase, the button, "log in sample" was clicked. The sample position was selected and this position corresponded with the position of our sample cup. The sample was given an ID. L292 fibroblast was selected as the cell type. The dilution factor was also selected if necessary. The results could be set up to be sent to an excel file. The details of the log were now available in the "autosampler queue".

Results:

L292 Harvest

The results from the cell harvest were simple. During the process the cells were adhered to the surface of the flask. They resembled a cobblestone design. The cells were ultimately placed into a centrifuge tube that was used for further experiment.

Cell Counting

Haemocytometer

Square 1- 18

Square 2- 24

Square 3- 19

Square 4- 23

Total cells - 84 cells

Average number of cells per square = 21cells

C = n/v;

When n = 21 cells, v = .0001 mL,

C = 21 X 104 cells/mL

Dilution Factor = 1

Vi-CELL Counter

The results from the Vi-CELL counter are as follows:

Total Cell Count = 392

Viability = 78.1%

Viable Cells/mL = .16 X 106 cells

Total Cells/mL = .21 X 106 cells

Comparison

Percent error = ( Absolute Value[ value 1 - value 2]/[value 1]) X 100

Percent error = 0%

Analysis of Cell Growth

Figure 4: The data that was given in the laboratory script [6]

Table 3: Data for plate 1 from table 1

Time (hours)

Average Cell Count

0

29226.67

6

29213

26

62560

48.5

148293.3

72

288160

96

547706.7

116

614640

136

634400

Graph Results

Cell seeding density: 30000 cells

Cell saturation density: 600000 cells

Doubling time: 22 hours

Sample Calculations (look at linear portion)

K = ln(N2/N1)/(t2-t1)

Doubling time = ln2/K

K = ln(148293.3/62560) = .0383

Doubling time = ln2/.0383 = 18 hours

Table 4: Data for plate 2 from table 1

Time (hours)

Average Cell Count

0

21520

6

22909

26

60266.67

48.5

146826.7

72

289973.3

96

529866.7

116

600586.7

136

599893.3

Graph Results

Cell seeding density: 21000 cells

Cell saturation density: 600000 cells

Doubling time: 25

Calculations

K = .03957

Doubling Time = 17.52

Table 5: Data for plate 3 from table 1

Time (hours)

Average Cell Count

0

19440

6

21657.33

26

60160

48.5

154533.3

72

280533.3

96

508453.3

116

583840

136

625840

Graph Results

Cell seeding density: 20000 cells

Cell saturation density: 600000 cells

Doubling time: 22

Calculations

K = .0419

Doubling Time = 16.53

Table 6: Data for plate 1 from table 2

Time (hours)

Average Cell Count

0

23379

6

29567

26

40481

48.5

57368

72

67106.33

96

104290.7

Graph Results

Cell seeding density: 24000

Cell saturation density: It did not saturate. The max value was just over 10000 cells.

Doubling time: 30 hours

Calculations

K =.0157

Doubling Time = 44 hours

Table 7: Data for plate 2 from table 2

Time (hours)

Average Cell Count

0

20214.33

6

23313

26

30340.33

48.5

48363.33

72

71207.33

96

69227

Graph Results

Cell seeding density: 21000 cells

Cell saturation density: 70000 cells

Doubling time: 40 hours

Calculations

K = .0131

Doubling time = 52 hours

Table 8: Data for plate 3 from table 2

Time (hours)

Average Cell Count

0

27551.67

6

22147

26

30874

48.5

45775

72

82053.33

96

92503.33

Graph Results

Cell seeding density: 27000 cells

Cell saturation density: 90000 cells

Doubling time: 45 hours

Calculations

K = .0175

Doubling Time = 39.6 hours

Summary of Results

The cells cultured in part one were healthy and viable. The counting methods in part two produced the same cell counts. In part three, the average growth rate for the cells in media A was .0399. The average doubling time was 17.36 hours. The average growth rate for the cells in media B was .0154. The average doubling time was 44.91 hours. The average saturation density for the cells in media A was 600000 cells. The average density for the cells in media B was 87000 cells.

Discussion:

The first part of the experiment consisted of basic cell culturing. The cells resembled a cobblestone appearance because they were healthy and viable. In order to be healthy and viable, the cells had to be cultured with caution and sterile techniques had to be used. The recorded observations showed that sterilization methods used were effective. The cells were washed with Calcium and Magnesium free DPBS. The solution did not contain calcium and magnesium because these two elements can block enzyme activity. More specifically, they could block trypsin activity. The cells were also regularly placed in EMEM +10% Foetal Bovine Serum. This serum is necessary for our cells because it provides nutrients and growth factors for cells to function normally. This keeps the cells healthy and viable.

The second part of the experiment consisted of counting the cells using two different methods. One method was using a haemocytometer. The other method was using a computer machine called the Vi-CELL counter. The results achieved were exactly the same. The number achieved was 21 X 104 cells/mL. This is unlikely to happen often. Most of the time, the values will just be very similar. They would be similar because both methods used were fairly reliable. The haemocytometer provided a grid for the most accurate way to count the cells by hand. The grid was used to calculate a certain amount of cells in one section. Calculations were used to determine the amount of cells per unit volume. The Vi-CELL counter uses algorithms to determine the number of cells in the sample. It can also calculate the number of cells that are dead and alive. It will report a viability number to show what percentages of the cells are alive. Both methods will count the cells but it is difficult to be completely accurate with either method. When using the haemocytometer, it is possible that the incorrect cell count was made for a certain square. It is possible that a cell was skipped or counted twice. It is also possible that cell debris was accidently counted as a cell. This could lead to various counts of cells. The Vi-CELL counter may also make errors. The counter may accidently count something that is not a cell. The Vi-CELL counter is probably more efficient for high concentrations of cells. There is less chance of miscounting the cells. Therefore, it will probably be more accurate as well.

The third part of the experiment consisted of analyzing cell count data. The two sets of cells were placed in different media conditions and the cell counts were recorded in tables. The saturation densities for the cells under media A were much higher (600,000 cells) than the saturation densities for the cells under media B (87,000). This could be due to higher growth rates for the cells under media A. There is a chance that the cells would continue to grow but there are no longer sufficient nutrients in the media to support larger numbers under media A. There is also a chance that there is no more room for growth in media A. The doubling time values and growth rates for the cells under media A were significantly faster than the doubling time values for the cells under media B. The average growth rate for media A cells was .0399 while the average doubling time was 17.36 hours. The average growth rate for media B cells was .0154 while the average doubling time was 44.91 hours. Both sets of cells began with similar seeded densities. The k value or the growth rate was much higher for the cells under media A. This could happen for several reasons. Media A could contain a higher concentration of CDK's. A higher concentration of CDK's could increase the rate of cell growth [3]. CDK's are used to regulate mitosis and the rest of cell growth [3]. Rather than having cells in the growth phases of G1 and G2 [4], CDK's could be used to propel the cells into mitosis and cytokinesis. This would increase the division rate and make the doubling time decrease. Another growth factor that could be present in the media is Fibroblast Growth Factor-2. This growth factor increases the rate of mitosis in mesenchymal stem cells [8]. Another possible factor that can affect the growth rate is temperature of environment. Increasing the temperature has been shown to increase enzymatic activity [5]. This could lead to an increase in enzymatic activity that contributes to cell division.

Conclusion:

In conclusion, it is safe to assume that sterile cell culturing techniques will result in healthy and viable cells. These cells can be observed under a microscope. Also, the two methods of cell counting used were both accurate. Both methods resulted in the same cell count but it is unlikely to happen this way every time. The values should be very similar though, but there is more likely to be error when using the haemocytometer. In the final part of the experiment, the cells in media A had higher growth rates than the cells in media B. Therefore, the cells in media A had smaller doubling times. The conditions in media A were more optimal for growth. The conditions in media A probably had more nutrients and more optimal temperatures for enzyme function.

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